Intracellular pathogens, such as Salmonella typhimurium, can survive within a macrophage and have evolved a number of mechanisms to resist the bacteriocidal mechanisms within these professional phagocytes. We have developed a simple in vitro assay and used it to identify Salmonella mutants that cannot survive within the macrophage. Roughly 1% of the Tn10 insertion mutants we have screened with this assay are not able to survive in the macrophage and all of these are avirulent as expected for a mutation in a known virulence mechanism. Secondary phenotypes have been assigned to 46 mutants identified in this screen. Many of the mutations are located in pathways that would be anticipated to be essential for survival in the macrophage (eg. sensitivity to oxidants) whereas others either have no secondary phenotypes or were not expected (sensitivity to complement components). Understanding of the defects in these mutants will contribute to the understanding of other clinically important and more complex intracellular pathogens.